1. Field of Invention
This invention relates to a novel ferulate esterase isolated from Lactobacillus fermentum, the amino acid and cDNA sequences of this novel ferulate esterase and its use in fermentation.
2. Description of the Prior Art
Lignocellulosic biomass materials are renewable, abundant and sustainable. In the most recent decade, lignocellulosic materials have been envisioned to replace petroleum resources for production of fuels and chemicals. Research focused on conversion of lignocellulosic materials to fuels and chemicals has become a global priority because of the limited petroleum supply. Based on chemical composition, lignocellulosic biomass contains three major polymers; cellulose (35-50%), hemicellulose (20-35%), and lignin (10-20%). Cellulose and hemicelluloses are covalently bounded through ester linkages to lignin via variety of hydroxycinnamic acids including ferulic, p-coumaric, sinapic and p-cafferic acid. The tight ester-ether bridges among the polysaccharides form the interwoven mesh-like plant cell wall structure that supports plant growth and development (Kroon and Williamson, J. Science of Food and Agriculture 79(3):355-361 (1999)). In nature, when plants die, these polymers and ester bridges are usually degraded and broken down slowly by complicated orchestrated action of cellulases, hemicellulases and other specific enzymes including ferulic acid esterases that are produced by microbes under suitable anaerobic environmental conditions.
Feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, resulting in the cleavage of the ester cross linkages. See MacKenzie, et al., Appl. Environ. Microbiol., 53(12):2835-2839 (1987); Rashamuse, et al., J. Applied Microbio., 103(5):1610-1620 (2007); and Kabel, et al., Appl. Environ. Microbiol., 77(16):5671-5681 (2011). These microbial ferulate esterases are also referred to hemicellulase accessory enzymes, because they are required to work together with xylanases and pectinases in degrading hemicelluloses of plant cell wall.
Ferulic acid esterases break down ester bonds between hydroxycinnamates and sugar (Crepin, et al., Appl. Microbiol. Biotech., 63(6):647-652 (2004); Topakas, et al., Process Biochemistry, 42(4):497-509 (2007); and Stewart, et al., Plant Physiology, 150(2):621-635 (2009)). The production of feruloyl esterase activities can be detected on agar plate such as MRS-ethyl ferulate plate, in which the main carbon source is substituted with 1% ethyl ferulate. The hydrolysis of ethyl ferulate by feruloyl esterase can be visualized as clear zones around the individual colonies. (Kin, et al., Appl. Environ. Microbiol., 75(15):5018-5024 (2009)). A number of Bacillus spp. predominantly B. subtilis strains, exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lactobacillus fermentum (NCFB 1751) shows the highest level of ferulic acid esterase activity. The enzyme is released from harvested cells by sonication and has pH and temperature optima of 6.5 and 30° C. respectively. (Szwajgier, et al., J. Instit. Brewing, 116(3):293-303 (2010); Szwajgier and Jakubczyk, Acta Scientiarum Polonorum, Technologia Alimentaria, 10(3):287-302 (2011)). Biochemical characterization and relative expression levels of multiple carbohydrate esterases of the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate has been performed (Kabel, et al. (2011)).
Because of the essential role ferulate esterase plays during biomass degradation and its use in the production of ferulic acid which is used in foods and in skin care products because of its antioxidant properties (see, Graf, E. Free Radic. Biol. Med. 13:435-448 (1992), it is useful to identify the cDNA and amino acid sequence of a highly active ferulate esterase and to express the highly active ferulate esterase in a heterologous bacteria and purify the enzyme.